NKT cells and SAP

Source: Chung B, Aoukaty A, Dutz J, Terhorst C, Tan R. Signaling lymphocytic

activation molecule-associated protein controls NKT cell functions. J Immunol.

2005 Mar 15;174(6):3153-7. PubMed PMID: 15749842.

 

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency disorder, which is caused by mutation in the gene Src homology 2 (SH2) domain containing gene 1A (SH2D1A). This gene encodes signaling lymphocyte activating molecule (SALM)-associated protein (SAP). There are many evidences that indicate that SAP plays a role in regulation of lymphocytes.

• The clinical course of XLP consists of uncontrolled proliferation of B and T cells.
  
• Studies have observed that SAP knock out (SAPKO) mice generate abnormally large virus specific CD8+ and CD4+ T cell following infection with LCMV.
  
• It has also been observed that SAP binds to intracellular domains of SLAM and B4, both of these are immuno-regulatory molecules, which are primarily expressed on T cells and NK cells, respectively.
  
• SAPKO mice are unable to generate memory B cell response.
  

Despite all these evidences about role of SAP in controlling lymphocyte proliferation, a role for SAP in NKT cells development and function, a type of regulatory cells, is not known. It has been previously reported that CD1d restricted NKT cells control EBV-specific lymphocyte proliferation [Ho et al., 2004 (CD4-CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion)]. This can be due to a possible link between NKT cell function and large T cell expansions seen in XLP patients following EBV infection.

In a beautiful paper published in 2005 from the labs of Dr. Rusung Tan, the investigators sought to determine the role of NKT cells in the immune dysregulation of SAPKO mice.

Their findings are briefly described below:

1. SAPKO mice completely lack CD1d restricted NKT cells.
   
2. When SAPKO mice were injected with potent NKT agonist alpha-GalCer, the mice did not develop or activate NKT cells that produce cytokines (IFN-gamma and IL-4).
   
3. When SAPKO mice were co-injected with OVA and alpha-GalCer, the mice did not show OVA specific CTL responses.
   
4. Patients with XLP did not have a population of CD1d restricted NKT cells in their peripheral blood.
   

All these findings indicated that SAP is critical for normal CD1d restricted NKT cell development and function.

 

Performance of experiments:

• SAPKO mice and wild type mice were taken and SAP gene expression was confirmed by PCR and immunoprecipitaion. Lymphocytes were stained with Abs to CD3 and NK1.1, these two molecules are the markers present on murine NKT cells, and flow-cytometric analyses were performed. The investigators observed that frequency of CD3+ NK1.1+ NKT cells from liver, thymus, spleen and lymph nodes was significantly low in SAPKO mice in comparison to wild type. An earlier study (Hammond et al., 2004) has shown that NK1.1 expression may not be the ideal marker for CD1d restricted NKT cells as many of the CD1d restricted cells fail to express NK1.1. Thus, Dr. Tan and his team used CD1d tetramer and Ab to TCRVbeta8 to stain lymphocytes from spleen, liver, thymus and lymph nodes from SAPKO and wild type mice. The data obtained confirmed that SAPKO mice lack CD1d-restrcited NKT cells.
  
• Up on activation NKT cells rapidly produce cytokines. The investigators thus hypothesized that since SAPKO mice lack CD1D-restricted NKT cells, they would not produce these cytokines upon activation. Thus, SAPKO and wild type mice were intra-peritoneally injected with alpha-GalCer and liver cells were isolates after 2 hrs. These cells were stained with CD1d tetramer to identify CD1d-restricted NKT cells and intracellular cytokine staining was performed using anti-IFN-gamma-allophycocyanin or anti-IL4-allophycocyanin. The authors observed no CD1d restricted NKT cells and no IFN-gamma or IL-4 production in SAPKO mice. In contrast, wild type mice produced significant amount of IFN-gamma and IL-4. These findings indicate that SAPKO mice are unable to mount a rapid NKT-induced cytokine response.
  
• A previously conducted study showed that NKT cells not only regulate LCMV-induce cytokine production, but they also controlled the magnitude of the cell-mediated immune response to an acute viral infection. Thus, NKT cells are important for generation and regulation of antigen specific T cells. Thus, to determine whether NKT cells are important for activation or proliferation of antigen specific T cells, the authors immunized SAPKO and wild type mice with vehicle (a control for alpha-GalCer) and OVA, CFA or alpha-GalCer. At day 14, PBMCs were isolated and stained with Kb-OVA254-267 tetramers and CD8 Ab to measure OVA-CTL expansion. Wild type mice showed significant CTL-expansion in comparison to SAPKO mice immunized with OVA and alpha-GalCer. These data indicate that the generation of Ag-specific CTL response is promoted by CD1d restricted NKT cells and SAPKO mice are unable to generate CTL in response to immunized Ag and NKT cell agonist.
  
• The scientists also took peripheral blood from two XLP patients, who had mutations in the second exon of SAP, and stained the PBMCs with CD1d tetramer and anti-CD3 Ab. They followed the same experiments in eight healthy individuals. The healthy controls showed significant percentage of CD1d-restricted NKT cells in comparison to XLP patients, indicating that CD1d-restricted NKT cells are absent in XLP patients.
  

 

Conclusions:

NKT cells act as a bridge between innate immune response and adaptive immune response by rapidly activating NK cells and helping in maturation of DCs. Thus, the authors hypothesized that in the absence of NKT cells, the immune response to virus is sub-optimal. Thus, viral replication occurs unchecked leading to formation of a chronic viral stage. It has been observed that boys with XLP disease show continuous viremia with EBV during the course of diseases. Such uncontrolled virus infection may lead to chronic stimulation of virus specific CTLs that are not efficient to clear virus-infected cells.

NKT cells suppress EBV-associated tumors

EBV, a first discovered human tumor virus, is considered to be one of the most successful human pathogen. It is involved in the development of many malignancies, such as Hodgkin's lymphoma and nasopharyngeal carcinoma in immunocompromised individuals. Majority of the individuals infected with EBV remain symptom free. Thus, it is believed that EBV has co-evolved with the human immune system to a peaceful co-existence.

It is known that natural killer T cells (NKT) are a special type of T cells that express NK lineage receptors including CD1d. They respond to glycolipid antigen presented by CD1d. CD1d is mainly expressed on antigen presenting cells (APCs, dendritic cells, macrophages and B cells). Thus, NKT cells primarily interact with APCs rather than tissues. Upon activation, these NKT cells release both Th1 and Th2 cytokines. However, it is present not known whether there are subsets of NKT that specialize in Th1 and Th2 cytokine production. Furthermore, how these cytokines lead to a regulated immune response in vivo is not known. Earlier studies have suggested that cytokine profile of NKT cells might be influenced by quality of TCR signal. The quality of TCR signal is in turn dependent on different antigens.

The present paper is from the lab of Dr. Yuling He, Wuhan University School of Medicine, China. In an earlier paper, the authors observed that CD8+ NKT cells drive syngenic T cells into a Th1-bias response to suppress EBV-associated malignancies and that IL-4-biased CD4+ NKT cells do not affect T cell cytotoxicity. In the present work, the authors sought to determine the mechanism of the EBV-induced CD8+ NKT cell response to EBV-associated malignancies.

Source: Xiao W, Li L, Zhou R, Xiao R, Wang Y, Ji X, Wu M, Wang L, Huang W, 

Zheng X, Tan X, Chen L, Xiong T, Xiong J, Jin Y, Tan J, He Y. EBV-induced 

human CD8(+) NKT cells synergise CD4(+) NKT cells suppressing EBV-associated

tumours upon induction of Th1-bias. Cell Mol Immunol. 2009 Oct;6(5):367-79.

PubMed PMID:19887050.

RESULTS:

1. Circulating mDC1cells from patients with EBV-associated malignancies impaired IFN-gamma production of CD8+ NKT cells: In another previous study, the authors observed that the frequencies of total and CD8+ NKT cells in PBMCs from Latent EBV infection (LEI) and control negative subjects (CN) were significantly higher than those from Hodgkin lymphoma (HL) patients and nasopharyngeal carcinoma (NPC) patients. To determine the interaction of CD8+NKT cells with APCs, the authors first determined frequencies and CD1d expression of circulating DCs, which included myeloid dendritic cells 1 and 2 (mDC1 and mDC2) and plasmacytoid dendritic cells (pDC), in PBMCs from NC and LEI subjects , and HL and NPC patients by staining with different markers, BDCA1 for mDC1, BDCA2 for pDC and BDCA3 for mDC2 and CD1d. The frequencies of mDC1 cells were comparable among HL and NPC patients, and LEI and NC subjects. CD1d was found to be predominant expressed on mDC1 cells than mDC2 and pDC cells. The expression level of CD1d was comparable on LEI and NPC subjects and HL and NPC patients. The authors next determined cytokine expression by CD4+ and CD8+ NKT cell lines. These cell lines were generated from PBMC from LEI subjects. Type 1 mDCs were purified from LEI and NC subjects and HL and NPC patients. These mDCs were than co-cultured with CD4+ or CD8+ NKT cell lines in the presence of alphaGalCer or alphaGalCer with CD1d mAb for 1 hr. The mDCs were then re-purified using staining with alpha-GalCer loaded CD1d tetramer. Then, the expression of IFN-gamma and IL-4 was assessed by Q-PCR. Both IFN-gamma and IL-4 were detected in CD4+ and CD8+ NKT cell lines cultured with mDCs from different groups of patients and subjects. The authors observed that both NKT cell lines from LEI and CN subjects highly expressed IFN-gamma upon stimulation with alphaGalCer. Although, not much effect was seen on IL-4 expression among various patients and subjects. Furthermore, using the same set of experiments the authors determined IFN-gamma and IL-4 expression by using ELISA. They observed that prestimulation with alphaGalCer caused significant increase in expression level of IFN-gamma in CD8+ NKT cell lines incubated with PBMCs from LEI and CN than from HL and NPC patients. IL-4 levels were in CD4+ NKT cells cultured with PBMCs from both patients and subjects. These results indicate that circulating mDC1 cells of HL and NPC patients cause a decrease in IFN-gamma production by CD8+ NKT cell line and have no effect on IL-4 production.
   
2. EBV induced CD8+ NKT cells promote a Th1 biased response that suppresses EBV-associated tumor cells in vitro: The authors observed in one of their early study that CD4+ NKT cells in PBMCs from HL and NPC patients and LEI and CN subjects produced similar low levels of IFN-gamma and high levels of IL-4, IL-13, IL-10 and TGF-beta1. In contrast, CD8+ NKT cells from control subjects produced expressed high levels of IFN-gamma and IL-2 but low levels of IL-4, IL-13, IL-10, and TGF-beta1. They also found that CD8+ NKT cells from LEI and CN subjects efficiently killed alpha-GalCer loaded EBV-associated HL and NPC tumor cell lines but not non-tumor cells. CD4+ from various patients and subjects either did not show such killing or showed very weak toxicity to both tumor and non-tumor cells. In the same study, the authors also observed that EBV-induced human NKT cells rapidly responded to EBV-associated tumor cells by secreting cytotoxic cytokines such as IL-10 and IFN-gamma. In the present study, the authors tried to verify whether this killing was specific, the authors first demonstrated present of EBV through the use of southern blot and QPCR in these cell lines. Thus, EBV-genes, LMP1, EBNA1, BZLF1, BALF2, and RAZ were examined in human EBV-associated tumor cell lines and non-tumor cell lines. The authors were able to detect these viral genes and their mRNA transcripts in tumor cell lines but not in non-tumor cell lines. The next task was to determine the mechanism by which NKT cells inhibit EBV-associated malignancies in vivo. Thus, the authors established hu-thym-SCID chimaeras. These chimaeras were implanted with human Hodgkin´s-derived EBV-associated B-cell lymphoma cell lines (LBC) or NPC. The animals were sacrificed 12 weeks post tumor implantation. Various organs including thyms, liver spleen and peripheral blood were collected and single cell suspensions were prepared. These cells were stimulated with alphaGalCer and cytokine expression (IL-4, IL-10 and IFN-gamma) was determined, using cytokine Ab, CD4 Ab, CD8 Ab and CD1d tetramer. The authors observed that a large number of CD8+ NKT cells expressed IFN-gamma and IL-2, but few cells expressed IL-4, IL-10 and IL-13. In contrast, CD4+ NKT cells expressed moderate levels of IL-4, IL-10 and IL-13 and low levels of IFN-gamma.
   
3. EBV- induced CD4+ NKT cells synergized with CD8+ NKT cells to promote a Th1 bias response against EBV-associated tumors: In their earlier work, the authors observed some kind of synergy between EBV-exposed thymic CD4+ and CD8+ NKT cells to suppress EBV-associated malignancies and prolong animal survival. To further investigate the mechanism of this synergistic effect, the SCID mice were adoptively transferred iv with different combinations of immune cells (CD4+/CD8+ NKT cells or both and CD3+CD56-Cd161- T cells) purified from EBV-exposed hum-thym-SCID chimaeras. After, three days these chimaeras were re-challenged with EBV. The scientists observed that in chimaeras transferred with CD4+ NKT cell alone, the re-challenge with EBV caused inhibition in proliferation of CD3+ T cell in comparison to chimaeras transferred with CD8+ NKT cell alone. Interesting, the chimaeras transferred with both CD4+ and CD8+ NKT cells showed a vigorous proliferation of CD3+ T cells and a high frequency of IFN-gamma producing cells.
   

To be continued……………….

ESCRT factors restrict mycobacterial growth

Mycobacterium tuberculosis is considered as one of the most successful pathogen. Some of the reasons that are responsible for its success include its ability to survive within the macrophages. But how does the bacteria survive intra-cellularly is not known. In the present paper, the authors have tried to address this question.

Source: Philips JA, Porto MC, Wang H, Rubin EJ, Perrimon N. ESCRT factors 

Restrict mycobacterial growth. Proc Natl Acad Sci U S A. 2008 Feb 26;105

(8):3070-5. Epub 2008 Feb 19. PubMed PMID: 18287038; 

PubMed Central PMCID: PMC2268586.

 

It is known that virulent mycobacterial are able to alter the maturation of phagosome inside the macrophages. They reside in a cellular compartment that resembles an early endosome. This endosome retains early endosome markers such as Rab5 and fails to recruit mature enodosomal markers such as Rab7.

In an earlier study, the authors performed a functional genomic screening to identify host factors, which can influence the uptake and growth of Mycobacteria. The authors used Drosophilla S2 cells, a macrophage like cell line that is open to RNAi and M. fortuitum, which like M. tuberculosis restricts phagosome fusion with lysosomes. M. fortuitum was able to induce the expression macrophage activated promoters (maps), which are the bacterial genes that are specifically induced when the bacteria grows intra-cellularly. Thus, by visualizing GFP (Green fluorescent proteins, proteins that produce free fluorescence when exposed to blue light) produced under the control of the map24 promoter, the authors were able to monitor the bacterial response to the intracellular environment. In another previous study, the authors identified 86 dsRNAs, which were able to diminish bacterial GFP production. As production of GFP reflects both the bacterial response to the phagosomal environment and bacterial growth, these dsRNAs can affect the infection by altering bacterial uptake, intra-cellular bacterial growth or induction from the map24 promoter.

In the present study, the authors further characterized three host cell activities Rab7, CG8743, and the ESCRT machinery. First to check the affect of dsRNAs, they used M. smegmatis, as it is difficult to measure intra-cellular growth of M. fortuitum. Thus, they examined two possibilities, if dsRNA treatment results in less intra-cellular growth of M. fortuitum and then it should have little effect on M. stegmatis because they do not grow in S2 cells. The other possibility was if the dsRNA altered phagosome environment and caused diminished map induction, then treatment with dsRNA should cause increase in permissiveness of phagosome for bacterial growth and M. stegmatis should grow. They treated S2 cells with dsRNA and then infected them with M. stegmatis. After two days, cells were examined by microscopy. They observed that four dsRNAs out of 86 strongly increased the percentage of heavily infected cells. These dsRNAs targeted Rab7, dVps28, CG8055 and CG8743. To confirm that bacterial growth was occurring intra-cellularly, they further blocked the bacterial uptake by using a dsRNA which targets Pes, a receptor required for entry of bacteria into the cell. The investigators observed that blocking did not cause any increase in bacterial growth and thus the results indicated that growth must be occurring intra-cellularly. Based on these results, the authors suggested that Rab7, dVps28, CG8055 and CG8743 might be playing important roles in restricting the intracellular growth of M. stegmatis.

Rab7 is a GTP binding protein and a marker for late enodosome. But the role of dVps28, CG8055 and CG8743 are not known. dVps28 and CG8055 are members of ESCRT machinery, which is made up of three proteins ESCRTI, ESCRTII and ESCRTIII. The authors thus, tested the effect of other dsRNAs that target additional ESCRT components on M. stegmatis growth. The authors observed that all dsRNAs, which robustly disrupt ESCRT machinery, caused increased growth of M. stegmatis and also altered the phagosome environment resulting in map24 and map49 induction by M. fortuitum and increased growth of M. stegmatis.

It is known that the ESCRT machinery acts on the membrane of the endosome. its function is to deliver ubiquitinated receptors into intraluminal vesicles of multivesicular bodies. These bodies are finally delivered to the lysosome for degradation. In Drosophila, a lack of ESCRT functioning causes accumulation of enlarged endosomes with accumulation of ubiquitin.

The authors next sought to determine whether ESCRT machinery acts directly on mycobacterial phagosome. They examined the localization of bacteria in ESCRT-depleted cells. S2 cells, which were depleted of dTsg101 and Vps28 (part of ESCRTI) were infected with M. stegmatis. After three hrs of infection, bacteria were found within the heavily ubiquinated vesicular compartments. These results showed that bacteria reside in the compartment on which ESCRT machinery acts. These results indicate that ESCRT machinery directly acts on the bacterial phagosome.

To test whether ESCRT machinery affects mycobacterial growth infection in mammalian macrophages, the authors depleted dTsg101 and Vps28 from murine macrophages RAW264.7 using SiRNA. These cells were then infected with M. fortuitum map24:GFP. The depletion caused less GFP production when cells were infected with M. fortuitum map24:GFP in comparison to controls. To further determine whether phagosome in ESCRT depleted cells was also less restrictive for mycobacterial growth, Raw264.7 cells were depleted of dTsg101 and Vps28 and infected with M. smegmatis hsp60::GFP. In control cells there was little growth 24 hrs post infection. In contrast, substantial growth was observed in depleted cells. Thus, the authors concluded that ESCRT machinery is required to restrict the growth of M. stegmatis in mammalian macrophages.

The diminished GFP production in cells depleted of ESCRT and infected with M. fortuitum map24:GFP is due to diminished expression and not due to decreased bacterial growth. To validate this, the authors used an M. fortuitum strain that expresses both red fluorescent protein (dsRed2) and GFP under control of two separate promoters. dsRed2 is under the control of msp12 promoter and is constitutively expressed. GFP is expressed under the control of map49, which is induced during intracellular growth. When ESCRTI depleted cells were infected with this bacteria, the authors observed diminished GFP expression as early as 3 hrs post infection. Examination of red fluorescence however, revealed that bacterial uptake was similar in both depleted and control cells and bacterial growth was similar or slightly increased in controls. These results suggested the phagosomal environment in ESCRT depleted cells was different from the non-depleted cells. The effect of this difference is such that the induction from the map24 and map49 promoters was decreased and bacterial growth is not efficiently restricted.

Evaluation of a novel Mycobacterium tuberculosis protein in the immunodiagnosis of TB

Source: Singh KK, Sharma N, Vargas D, Liu Z, Belisle JT, Potharaju V,

 Wanchu A, Behera D, Laal S. Peptides of a novel Mycobacterium tuberculosis

-specific cell wall protein for immunodiagnosis of tuberculosis. J Infect Dis.

2009 Aug15;200(4):571-81

 

Mycobacterium tuberculosis is an opportunistic pathogen that causes nearly 2-3 million deaths each year. One of the common methods of diagnosis is by microscopic identification of acid fast bacilli in sputum samples. However, the procedure is tedious, and requires multiple samples. Also, the success rate of correct diagnosis has been reported to be 30-60%. Thus, rapid, point-of-care diagnostic tests are required. Currently, there is no sero-diagnostic test that provides accurate diagnosis of M. tuberculosis infection. Many studies are going on to discover and identify new proteins of this pathogen, which can then be used to develop sero-diagnostic tests.

The authors of this paper previously identified a proline-threonine repetitive protein (PTRP; Rv0538) in aerosol infected rabbits. Earlier studies have shown that it is present in the cell wall. The authors of this paper also observed in their previous study that this protein does not have a homolog in M. avium and M. leprae.

Thus, the previous findings of immunogenicity of PRTP in rabbits, cell wall localization of this protein and its absence in the M. avium and M. leprae, made the authors to further characterize this protein and study its diagnostic potential.

The results obtained are discussed below:

1. ptrp is specific for M. tuberculosis (complex): The authors performed a protein BLAST analysis of PTRP. The results showed nearly 100% identity with a protein in M. tuberculosis CDC1551, M. tuberculosis F11, M. tuberculosis C, M. tuberculosis H37Ra, M. bovis AF2122/97 and M. bovis BCG strain. No such identity was observed in pathogenic non-tuberculous mycobacterial species. Nucleotide BLAST analysis of ptrp gene identified nearly 100% identical genes in Mycobacterium tuberculosis complex species and clinical isolates but not in the non-tuberculous mycobacterial species. The specificity of ptrp to the M. tuberculosis complex was further confirmed by Southern hybridization.
   
2. 
   Expression and purification of rPTRP: The PTRP protein was expressed and purified and its molecular weight was nearly 52kDa. Its identity was confirmed by sequencing.
   
3. PTRP is a cell wall protein of M. tuberculosis: To confirm the localization of this protein, sub-cellular protein fractions of M. tuberculosis were separated on a Western Blot and probed with pre-immune IgG, anti-PTRP IgG and anti-MS (malate synthase) IgG antibodies. Anti-PTRP antibodies identified a nearly 52KDa protein in total cell wall (TCW), SDS extracted cell wall (SDS-CW) of M. tuberculosis. Some weak reactivity was also observed in whole cell lysate (WCL), but no similar protein was observed in culture filtrates. It has been shown through earlier studies that malate synthase (MS) of M. tuberculosis is anchored to the cell wall. Thus, to estimate the relative abundance of PTRP in different cell preparations, the authors used anti-malate synthase (MS) IgG. With quantity of MS in each fraction considered to be 100%, WCLs were found to contain less PTRP than the TCW and SDS-CW. Finally, the presence of PTRP on cell wall was confirmed by immuno-electron microscopy.
   
4. Immunogenicity of PTRP in patients with TB: To determine the immunogenicity of PTRP in patients with TB, the reactivity of rPTRP was examined with serum specimens from patients with TB and healthy controls. The investigators observed that serum antibodies from 4 of the 6 HIV-negative TB positive and 5 of 6 HIV positive and TB positive patients displayed strong reactivity with rPTRP. None of the 6 HIV negative PPD positive, 6 HIV negative PPD negative or 6 HIV positive, TB negative patients showed any reactivity with rPDRP.
   
5. Identification of immunogenic epitopes of PTRP: The authors next checked the diagnostic potential of PTRP. Thus, they synthesized fifty-four overlapping peptides (20 aa in length with a 10-aa overlap; PT1–PT54) covering the entire PTRP sequence, each linked with a biotin residue at the N-terminal. Out of these 54, 53 peptides were tested for their reactivity with serum specimens from 13 PPD-negative and 23 PPD-positive healthy control subjects. No significant difference was observed in the reactivity of 49 peptides. No control serum tested positive with 22 of 53 peptides. The remaining peptides were recognized by the antibodies in only one or two serum specimens. When the 53 peptides were tested for their reactivity with serum specimens from HIV negative, TB positive patients, 16 of 53 peptides were recognized by antibodies from more than 40% of the patients with TB. Of these 16 peptides, 12 were reproducibly recognized by antibodies in specimens from more than 40% of patients with TB. Out of these 12 peptides, 4 were recognized by antibodies from specimens from more than 50% of patients with TB. These are PT9, PT13, PT40 and PT41.
   
6. Reactivity of immunodominant PTRP peptides with serum specimens from other classes of patients with TB: The reactivity of 4 immunodominant peptides was checked with serum specimens from different groups of patients and controls. 25-45% of smear negative, HIV negative, TB positive patients' serum specimens recognized these 4 immunodominant peptides. These peptides were also recognized by serum specimens from 60-76% of the HIV positive TB positive patients.
   

Concluding remarks:

In authors words: "These results demonstrate that immunodominant epitopes of carefully selected M. tuberculosis specific proteins can be used to devise a simple peptide based serodiagnostic test for TB."

Type IV secretion system in Helicobacter pylori: Cag3 function

Source: Pinto-Santini DM, Salama NR. Cag3 is a novel essential component of the Helicobacter pylori Cag type IV secretion system outer membrane subcomplex. J Bacteriol. 2009 Dec;191(23):7343-52. Epub 2009 Oct 2. PubMed PMID: 19801411; PubMed Central PMCID: PMC2786551.

 

Bacteria translocate or transfer effector molecules, for examples proteins, toxins, DNA and enzymes, from the interior to the exterior through secretion. The cell wall of bacteria plays an important role in the secretion. The structure of cell wall of Gram negative bacteria (GNB) is more complex than Gram positive bacteria. Thus, secretion is also more complex in GNBs. According to the literature there are at least six specialized secretion systems in GNBs. These are type I secretion system (T1SS), type II secretion system (T2SS), type III secretion system (T3SS), type IV secretion system (T4SS), type V secretion system (T5SS) and twin arginine translocation.

T4SSs are known to be involved in horizontal DNA transfer to other bacteria and eukaryotic cells, in uptake or release of DNA from/into the extracellular environment, in the secretion of toxins and in the secretion of virulence factors into host cells. It is similar to conjugation system of bacteria and can transfer both proteins and DNA. A lot of research has been going on to define the structure of these T4SS, to identify the molecules which are translocated and effect and mechanism of action of these effector molecules on the host cells. This information is essential to understand the pathogenecity of these bacteria and to design strategies to combat bacterial infections.

Helicobacter pylori is a GNB and it infects 50% of the world population. The infection can lead to chronic gastric and peptic ulcer disease, gastric carcinoma and mucosa associated lymphoid tissue lymphoma. However, only a small percentage of infected people suffer from H. pylori associated illnesses. Earlier studies have established that infection with strains harboring the cag pathogenecity island (PAI) leads to a much higher risk for development of severe illnesses. Cag PAI is a 40kb stretch of DNA that encodes 27 putative proteins. The T4SS VirB/D4 of plant pathogen Agrobacterium tumefaciens has been studied in great detail. Several of the proteins encoded by cag PAI share sequence similarity with components of A. tumefaciens VirB/D4 T4SS. The activity of cag PAI encoded T4SS is responsible for translocation of effector molecule CagA into the host cells and subsequent induction of proinflammatory cytokines.

In this paper, the authors have characterized the biochemical role of a H. pylori specific gene, HP0522/cag3, in Cag T4SS. A previous study has shown that Cag3 could interact with HpVirB8, HpVirB7, CagM and CagG. To understand the molecular basis of Cag3 function in T4SS, the authors investigated the subcellular localization of Cag3 protein as well as the interaction that this protein established with other proteins in bacterial cells.

A brief summary of major results obtained is as follows:

Results:

• cag3 is essential for Cag T4SS function: The CagT4SS is responsible for phosphorylation of CagA and induction of IL-8 secretion. Thus, to evaluate whether cag3 is essential for Cag T4SS function, the authors used a null cag3 mutant, a null cag3 mutant complemented by expression of gene at an unrelated locus rdxA and incubated the mutants as well as the wild strains with AGS cells and determined CagA phosphorylation and secretion of IL-8. The mutant strain was unable to translocate CagA as there was lack of phosphorylation of CagA and was unable to induce IL-8 secretion. However, in case of strains complemented with expression of cag3 at rdxA locus, both the CagA phosphorylation and induction of Il-8 secretion were observed.
  
• Cag3 is a membrane associated protein: To determine the location of Cag3, the investigators fractionated H. pylori cells and performed immunoblotting of each fraction with antibodies to Cag3. Cag3 was found enriched in the membrane associated fractions.
  
• Cag3 co-purifies with predicted T4S compartments: To identify the proteins that interact with Cag3, the authors used affinity purification followed by mass spectrometry. Total extracts from wild type and mutant strains were loaded onto anti-Cag3 affinity columns. The eluates were evaluated for their protein profiles. A number of proteins co-purified specifically with Cag3, i.e. they were absent in eluates of mutant cag3 extracts. Mass spectrometry analysis identified HpVirB7, HpVirD4, CagD and Cag1 as the most specific interactors. Other proteins identified were HpVirB4, HpVirB11, hpVirB10, HpVirB9, HpVirB2 and CagM.
  
• Cag3 interacts with predicted lipoprotein HpVirB7 independent of CagM: Previous studies have shown that HpVirB7 is a part of outer membrane subcomplex and previous studies have also suggested that Cag3 and HpVirB7 can interact. Thus, the authors further looked at the interaction of Cag3 and HpVirB7. For this, whole cell lysates from cag3 mutant strains and wild type strains (expressing HpVirB7-3XFLAG) were immuno-precipitated with anti-FLAG and anti-Cag3 antibodies. The authors observed that anti-FLAG antibodies were able to co-immuno-precipitate Cag3 in a wild type strain but not in the mutant strain. Similarly, anti-Cag3 antibodies were able to co-immuno-precipitate HpVirB7-3XFLAG from the wild type strain and not from the cag3 mutant strain. These observations confirm that Cag3 interacts with HpVirB7. The authors next sought to determine whether the Cag3 and HpVirB7 interaction was CagM dependent. The authors observed that anti-Cag3 antibodies were able to co-immuno-precipitate HpVirB7-3XFLAG in a cagM mutant. These observations indicate that Cag3 can interact with HpVirB7 independently of CagM.
  
• Cag3 and HpVirB7 fractionate in a high molecular weight complex: To further determine the size of Cag3 complexes in the cell, the authors fractionated whole cell wild type extracts and determined the presence of Cag3 and HpVirB7-3XFLAG in each fraction by immunoblotting with anti-Cag3 or anti-FLAG antibodies. Cag3 was precipitated in two peaks; one peak overlapped with the void volume while other peak corresponded to a 150KDa peak. The size predicted for Cag3 monomer is 55KDa. Thus, these results indicate that Cag3 exists in cell as part of two pools. The lower molecular mass form may represent the soluble form, while the higher molecular mass form may represent the Cag3 membrane associated complex.
  
• Protein cross linking reveals HpVirB7-CagM and Cag3-CagM interactions: H. pylori cells (wild type expressing HpVirB7-3XFLAG, mutant HpVirB7, mutant cag3, mutant cag3 complemented with expression of gene at rdxA, and mutant cagM) were cross-lined in vivo with formaldehyde. These cross linked whole cell extracts were fractionated and by SDS-PAGE and immunoblotted with ant-Cag3 or anti-FLAG antibodies. On immunoblotting with anti-Cag3 antibody, monomer sized band as well as several high molecular mass Cag3 containing complexes were observed. These complexes were present even in the HpVirB7 mutants, indicating that HpVirB7 was not present in these complexes. Surprisingly, one complexe was absent in cagM mutant indicating that this complex which was composed of Cag3 and CagM. In contrast, on immunoblotting with anti-FLAG antibody, only two bands were observed. One band corresponded to HpVirB7-3XFLAG while other band was absent in cagM mutant strains, indicating that a dimer containing HpVirB7 and CagM.
  
• Cag3 and HpVirB7 promote each other's stability: The mutant and wild type strains were metabolically labeled with Trans35S-label cys-Met during liquid culture growth for a 30 minutes pulse. After the pulse, the two aliquots from each culture were immuno-precipitated with anti-FLAG or anti-CagA antibody. HpVirB7-3XFLAG was less stable in the cag3 mutant strain background compared to the wild type. Cag3 appeared less stable in the absence of HpVirB7, HpVirB9 or HpVirB10.
  

 

 

Preferential translation of Vesicular Stomatitis Virus (VSV) mRNAs

Source:

Whitlow ZW, Connor JH, Lyles DS. New mRNAs are preferentially translated

during vesicular stomatitis virus infection. J Virol. 2008 Mar;82(5):2286-94.

Epub 2007 Dec 19. PubMed PMID: 18094194; PubMed Central PMCID: PMC2258916.

 

 

Introduction:

Vesicular Stomatitis Virus (VSV) is a virus that belongs to the same family of viruses to which Rabies virus belong, i.e. Rhabdoviridae. The infections occur in two steps, cytolytic infections of the mammals and transmission by insects. It is a negative sense single stranded RNA virus. The virus replicates inside the cytoplasm of infected cells and viral mRNAs are transcribed from the viral genome by viral RNA dependent RNA polymerase (RDRP). These mRNAs are quite similar in structure to host mRNAs. The translation of viral mRNA is dependent on the host cell machinery. Most of the viruses have developed mechanisms that inhibit host protein synthesis while viral mRNAs are preferentially translated. A lot of research has been going on to understand the mechanisms behind preferential translation of viral mRNAs. Knowledge of these mechanisms is essential for understanding viral replication.

Earlier studies on VSV have shown that the matrix (M) protein inhibits host gene expression at multiple levels. These include transcription, transport of mRNA into the cytoplasm, and translation. Studies have also shown that in cells infected with VSV, viral protein synthesis increases while host protein synthesis decreases. Thus, in the present study, the authors sought to determine why viral mRNAs are translated during the time that translation of host mRNAs is inhibited.

Results:

• mRNA transfected during infection is resistant VSV-induced translation inhibition, while mRNA transfected before infection is translationally inhibited by VSV : As it is known that during VSV infection, there is inhibition of host transcription and mRNA transport, which prevents new host mRNAs from reaching the cytoplasm and viral transcription is the primary source of mRNA in the cytoplasm. At the same time, there is inhibition of host translation and the predominance of viral translation. Based on these observations the authors speculated that the timing of transcription may be involved in the control of translation in VSV infected cells. They speculated that most recently synthesized mRNA might be translated preferentially. Thus to evaluate this hypothesis, the authors examined whether the time of appearance of mRNA in the cytoplasm relative to the time of infection was involved in controlling translation by trasfecting HELA cells with in-vitro transcribed EGFP reporter mRNAs at different time points relative to time of infection. Thus, HELA cells were transfected with SP6-EGFP mRNA and translation rates at various time points after infection were determined by pulse labeling with S35methionine. EGFP synthesis was determined by immunoprecipitation and measurement of labeled EGFP by SDS-PAGE and phosphorimaging. They observed that the minimum time required for transfected SP6-EGFP mRNA to be translated optimally is around 4h. In the next set of experiments, HeLa cells were trasfected with SP6-EGFP mRNA 22hrs before infection, 4h before infection or 1h after infection with a recombinant VSV expressing a wild type M protein. They observed that in recombinant wild type virus infected cells that were transfected 22 or 4h before infection, EGFP was synthesized at much lower rates in comparison to mock-infected cells. In cells in which SP6-EFGP mRNA was transfected at 1h post infection, EGFP was synthesized at much higher rates than in mock infected cells. These results clearly show that mRNA transfected during infection is resistant to VSV-induced inhibition, while mRNA transfected before infection is translationally inhibited by VSV.
  
• Transfection has no effect on viral protein synthesis: The authors also analyzed total cellular protein synthesis by SDS-PAGE and phosphorimaging in total cell lysates of cells that were transfected 22h before infection or of untrasfected cells that were subsequently mock infected or infected with rwt virus. Transfection of SP6-EGFP mRNA slightly reduced levels of total protein synthesis in comparison to control and this reduction was independent of time of infection. They observed that Inhibition of total protein synthesis by rwt virus was similar for all three times of transfection.
  
• mRNAs introduced into the cytoplasm during infection or close to the time of infection are resistant to VSV induced inhibition of translation: Furthermore, rates of SP6-EGFP mRNA protein synthesis and total host protein synthesis relative to those in mock infected cells were determined. When cells were transfected 22h before infection, the production of SP6-EGFP was reduced to less than half of that in mock-infected cells. There was a similar reduction in total host protein synthesis. When cells were transfected 4h before infection, the translation was inhibited but not as much as observed in case of cells transfected 22h before infection. In contrast, when cells were transfected 1h after infection, EGFP was translated almost two fold better in cells infected with rwt virus than in mock-infected cells. Also, the translation rate of EGFP relative to mock infected cells was significantly different from translation rate of total host protein synthesis in this case.
  
• The ability of VSV to inhibit host translation is separated from its ability to promote translation of viral mRNAs: To prove this point the authors determined translation efficiencies of host-derived and virus-derived EGFP mRNAs at different time points after infection with virus containing wild type or mutant M protein (not able to inhibit host protein synthesis). For this they infected HeLA-EGFP (host derived mRNA) with mock or rwt or M51R-M virus and HeLa cells with mock or rwt-EGFP or M51R-M-EGFP virus (virus-derived mRNA). EGFP was analyzed at 4, 8 or 12h post infection. The data obtained from these experiments showed that EGFP translation from HeLa-EGFP cells was inhibited to a greater extent following infection with rwt virus than with rM51R-M virus. In case of HeLa cells, infection with rwt-EFGP virus caused increase in EGFP synthesis from 4 to 8h but decrease by 12h post infection. Infection with rM51R-M-EGFP caused EGFP synthesis similar to that observed in HeLa cells infected with rwt-EGFP virus, although EGFP remained elevated at 12h post infection. The authors further determined the mRNA levels by Northern Blotting. In HeLa-EGFP cells infected with rwt virus, the level of EGFP mRNA was not significantly different from the level observed in mock infected cells until 12h post infection. After 12h, EGFP mRNA level in rwt-infected cells was reduced to 0.28 relative to mock infected cells. In HeLa-EGFP cells infected with rM51R-M virus, no difference in EGFP mRNA levels was observed in comparison to control at any time points. In HeLa cells infected with rwt-EGFP virus, the EGFP mRNA levels were 10 to 12 times to that observed in mock infected cells and were consistent throughout the time course. Translation efficiencies were determined by dividing the EGFP translation rates by the EGFP mRNA levels. Infection of HeLa-EGFP cells by rwt virus caused dramatic reduction in translation efficiency in comparison t controls 12 h post infection. Infection with rM51R-M also reduced translation efficiency, although to a lesser extent in these cells. In contrast, infection with of HeLa cells with rwt-EGFP and rM51R-M-EGFP, translation efficiency was found to be increased in comparison to controls. These results indicate that inhibition of host translation does not affect the translation efficiency of viral mRNAs before 8 h post infection and thus the ability of VSV to inhibit host translation is separated from its ability to promote viral translation.
  

Important Findings:

1. The authors show that newly appearing mRNAs are resistant to VSV induced inhibition of translation
   
2. Efficient translation of viral mRNAs or other newly appearing mRNAs is not dependent on inhibition of host translation
   
3. The increase in translational efficiency of viral mRNAs between 4 to 8h post infection is not due to inherent changes in mRNA translatability over time
   

Future Aspects: It seems that VSV and perhaps other viruses exploit the phenomena of preference of newly synthesized mRNA by translation machinery. The mechanisms that are responsible for the preferential translation of newly transcribed mRNAs in VSV infected cells are not known. During certain type of stress responses, preexisting mRNAs are shuttled into stress granules, while newly synthesized mRNAs are preferentially translated. These similarities can be exploited to understand the mechanisms responsible for such preferential translation.

HIV Selectest enzyme immunoassay and rapid test: ability to detect sero-conversion following HIV-1 infection

Most of the currently tested HIV vaccines contain multiple viral components. Due to this many vaccine recipients give positive results when tested for HIV sero-detection. Thus, to differentiate between vaccines induced antibodies and sero-conversion due to true HIV infection, the group of Dr. Hana Golding at Division of viral products, Center for Biologics Evaluation and Research (CBER), FDA, Bathesda, US, developed a new HIV immunoassay termed HIV Selectest. To develop this test, they identified conserved sequences in Env gp41 and Gag p6. These sequences are recognized soon after the infection but are not included in most HIV vaccine candidates. In that study, the investigators observed that HIV-Selectest could become an important differential diagnostic tool for HIV vaccine trials, blood banks and population screening worldwide.

In the present study, the investigators of Dr. Golding's lab together with investigators from various research institutes in Africa and USA, developed a rapid version of the HIV selectest in order to facilitate point of care testing during vaccine trials.

The authors obtained serial samples from following multiple cohorts of men and women from the United States and Africa:

• Plasma donors who acquired HIV-1 in the US (Center for HIV/AIDS Vaccine Immunology, CHAVI, clade B, predominantly males)
  
• High risk subjects identified with having acute HIV-1 infections in US sites participating in the Acute Infection and Early Disease Research Program (AIEDRP, clade B, predominantly males)
  
• Acutely infected subjects identified in Africa by the center for AIDS program of Research in South Africa (CAPRISA, clade C, mostly women)
  
• Subjects participating in the Zambia-Emory HIV research project (ZEHRP, clade C)
  
• Early post-infection time points in men enrolled in the US multicenter AIDS cohort study (MACS) and
  
• Women participating in the women's interagency HIV study (WIHS)
  

Results:

Of the 87 sero-conversion plasma donors tested, only 45 reached sero-conversion when HIV-selectest was performed on them. Of these 45 panels, concordant results with Abbott third generation tests and HIV-Selectest test were seen in 18 panels. The Abbott test detected positive results prior to Selectest in 20 of the 45 panels. The Selectest detected positive prior to Abbott test in 7 of 45 panels. On an average, the Selectest detected anti-HIV antibodies 1.6 days after the commercial Abbott test. In the other panels (AIEDRP, MACS, WIHS) the sensitivity of HIV Selectest ranged from 98.7 to 100% and from 93.8 to 98.3% in men and women, respectively. In case of clade C infections (CAPRISA), the HIV-Selectest was positive in later than one sample after sero-conversion. Taken together all these data indicated that the sensitivity of HIV-Selectest EIA was comparable to commercially available tests which are used in the HIV screening algorithm in many countries. To further evaluate and compare the sensitivity of HIV-Selectest rapid test and EIA with the commercially available third generation test, authors examined sero-conversion panels from Sera care Bioservices, Inc. In the case of panel PRB917, the Abbott HIV-1/2-O test was reactive on day 28, while the HIV-selectest EIA and rapid tests were positive on day 14.

Source: Khurana S, Norris PJ, Busch MP, Haynes BF, Park S, Sasono P,

 Mlisana K, Salim AK, Hecht FM, Mulenga J, Chomba E, Hunter E, Allen

 S, Nemo G, Rodriguez-Chavez IR; Women's Interagency HIV Study 

Collaborative Study Group, Margolick JB; Multicenter AIDS Cohort Study

 (MACS), Golding H. HIV-Selectest enzyme immunoassay and rapid test: 

ability to detect seroconversion following HIV-1 infection. 

J Clin Microbiol. 2010 Jan;48(1):281-5. Epub 2009 Nov 11. 

PubMed PMID: 19906903;

PubMed Central PMCID: PMC2812287.

 

A vital role of IL-21 in control of a chronic viral infection

Source: Yi JS, Du M, Zajac AJ. A vital role for interleukin-21 in the control of a

chronic viral infection. Science. 2009 Jun 19;324(5934):1572-6. Epub 2009 May 14.

PubMed PMID: 19443735; PubMed Central PMCID: PMC2736049.

 

CD4+ and CD8+ T cell responses play a major role in body's fight against viral infections. Earlier studies have shown that during the initial phases of many viral infections, CD8+ T cell responses are induced but they are failed to attain elaborate effector functions. The reason for this is not known. Although, it is known that in the absence of CD4+ T cell help, CD8+ T cell functions are impaired. CD4+ T cells are the primary producers of Interleukin-21 (IL-21). IL-21 has many functions; some of which are inducing the development of Th17 and follicular helper T cells (Tfh). As it has been shown that CD4+ help is essential for effective CD8+ cell response, the authors of this paper studied the role of IL-21 in CD8+ T cell responses to viral infections.

This paper is from the laboratory of Dr. Allan J Zajac, Department of microbiology, University of Alabama at Birmingham, US. In an earlier study, the authors observed a marked difference in the induction of IL-21+CD4+ T cells following acute LCMV Armstrong (arm) and chronic LCMV clone-13 (Cl-13) infections in C57BL/6 mice. They observed that although both groups of mice showed polyclonal virus specific CD4+IL21+ T cells by eight day of infection, this response was 7.8 times lower in mice infected with Cl-13.

This pronounced IL21+ CD4+ response in LCMV-Arm infected mice let the investigators to investigate whether IL-21 influences the generation of germinal center (GC) B cells, Tfh cells and antibody response. They observed that the percentage and number of GC B cells, Tfh T cells as well as LCMV-specific antibody titers were similar in IL21+/+ and IL21-/- mice, following 8-9 days of LCMV-arm infection. These results suggest that acute LCMV infection can trigger these responses even in the absence of IL21. Next, the authors analyzed responses of IL21-/-, Il21+/- and Il21+/+ mice to LCMV-Cl-13 infection. They observed that eight days after infection, the magnitude of anti-viral CD4+ and CD8+ T cell responses and the frequency of interferon gamma producing CD8+ T cells were similar among the three groups of mice. However, they observed differences in their functional quality. Both the percentages and absolute numbers of polyfunctional, IL-2 producing CD8+ T cells were reduced in IL21-/- and IL21-/+ mice. A similar trend for TNF-alpha production was observed. These results indicate that IL-21 deficiency results in impaired polyfunctional effector CD8+ T cell responses during initial phase of LCMV infection.

Furthermore, the authors checked the viral loads in these three groups of mice following LCMV-Cl-13 infection. They found that viral titers were highest in IL21-/- mice. These data suggest that IL-21 is critical for control of chronic viral infections. When the authors examined the anti-viral CD8+ T cell responses at later stages of LCMV-Cl13 infection, they found that by 136 day, infection was under control in IL21+/+ mice. IFN-gamma, IL-2 and TNF-alpha production was detectable by CD8+ T cells and these CD8+ T cells were CD43intermediate, programmed death -1 (PD-1) low. This phenotype is similar to resting memory T cells. In contrast, IL-2, IFN-gamma, and TNF-alpha production were highly reduced by CD8+ T cells in IL21-/- mice and these cells were CD43high, PD-1 high, a phenotype which is hallmark of exhaustion that develops in chronically infected hosts. These results clearly indicate that the absence of IL-21 production results in a failure to contain the infection and a substantial reduction in antiviral CD8+ T cell functions.

The next objective was to determine whether IL-21 acts directly to promote and sustain CD8+ T cell responses. Thus, the cohorts of IL21r+/+ / IL21r-/- (experimental) and IL21r+/+/IL21r+/+ (control) mixed bone-marrow chimeras were infected with LCMV-Cl-13 and CD8+ T cell responses were evaluated over time. The authors observed that both the IL21r+/+ and IL21r-/- CD8+ T cells were detectable in circulation following day 8 of infection. In contrast at 16^th day following infection, a preferential and rapid loss of IL21r-/- CD8+ T cells was observed. These data indicate that IL-21 is directly required for supporting and maintaining anti-viral CD8+ T cells during chronic viral infection.

To evaluate whether the addition of IL-21 enhanced anti-viral CD8+ T cells, the authors also performed experiments in which IL21 was administered to LCMV-Cl-13 infected CD4-/- mice. They found that daily injection of recombinant Il-21 to these mice, enhanced the functional quality of anti-viral CD8+ T cells.

Conclusions: The findings presented in this paper are quite interesting and throw new light into the role of IL-21 in successful control of infection. the authors showed that IL21 plays important role in induction of an effector CD8+ T cell response.

In authors words, " we anticipate that the cautious development of approaches to modulate the levels of IL-21, or regulate the induction of cellular subsets that generate IL-21, will provide new therapeutic opportunities to improve immunity to diseases that require CD8+ T cell responses to be controlled, such as chronic viral infection and tumors".